Mechanism of formation, spectrum and reactivity of half-reduced eight-iron Clostridium pasteurianum ferredoxin in pulse-radiolysis studies and the non-co-operativity of the four-iron clusters.
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ABSTRACT:
SUBMITTER: Butler J
PROVIDER: S-EPMC1161580 | biostudies-other | 1979 Nov
REPOSITORIES: biostudies-other
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Project description:1. Mössbauer spectra of both redox states of the eight-iron ferredoxin from Clostridium pasteurianum were observed over a range of temperatures and in magnetic fields. 2. At high temperatures (77 degrees K and above) the spectra of both states consist essentially of the superposition of two or more closely similar doublets. 3. The average chemical shift for the oxidized protein leads to the proposal that each of the two four-iron active centres consists formally of two Fe(3+) and two Fe(2+) atoms. 4. The average chemical shift and quadrupole splitting increase on reduction, consistent with there being one Fe(3+) and three Fe(2+) atoms per centre in the reduced molecule. 5. The spectral changes on reduction show that all the iron atoms are affected when one electron is added to each four-iron centre. 6. No separate Fe(3+) and Fe(2+) spectra were observed (as they were, for instance, in the reduced two-iron plant ferredoxins) suggesting that the d electrons are not localized on particular atoms, but are shared approximately equally by all four atoms in the four-iron centres. 7. At low temperatures (4 degrees K and below) no magnetic hyperfine interaction was observed in the oxidized protein even in an applied magnetic field, confirming the non-magnetic nature of the molecule in the oxidized state, and suggesting that the four iron atoms in each centre are antiferromagnetically coupled together to give zero spin. 8. Magnetic hyperfine interaction was observed in the reduced protein at low temperatures, and showed that all the iron atoms were magnetic. This demonstrates that one electron goes to each centre on reduction. 9. On application of a large magnetic field to the reduced protein at low temperatures, both positive and negative hyperfine fields were shown to be present, thus directly showing that antiferromagnetic coupling exists between the iron atoms in the reduced state.
| S-EPMC1166255 | biostudies-other
Project description:Both the oxidized and reduced forms of Hipip (high-potential iron--sulphur protein) are reduced (approx. 30% yields) by eaq.- in a single-stage process, rate constants 1.7 x 10(10) and 1.8 x 10(10) M-1 . s-1 respectively, at 25 degrees C, pH 7.0 (5 mM-phosphate). Super-reduced Hipip, which is formed in the latter case, has a spectrum which closely resembles that of reduced ferredoxin, i.e. Fe4S4 (SR)4(3-) clusters. The spectrum is stable over 2 s periods investigated. Super-reduced Hipip is reoxidized with O2, rate constant 4.8 x 10(6) M-1 . s-1 at 25 degrees C.
| S-EPMC1162045 | biostudies-other
Project description:Resonance Raman spectra of native 2[4Fe-4S] ferredoxin from Clostridium pasteurianum and of its selenium-substituted analogue 2[4Fe-4Se] are compared. The experimental conditions used in this study included low temperature (approx. 25K) and the absence of a glass sample cell, thus ensuring high signal-to-noise ratios. The spectra of the 2[4Fe-4S] and 2[4Fe-4Se] ferredoxins display similar numbers of bands, but the resonance Raman patterns differ largely, except for two bands observed at 353 cm-1 and 365 cm-1 in spectra of the native ferredoxin, which are only moderately shifted upon S----Se substitution and are attributed to Fe-S(cysteine) stretching modes. The activities of the latter modes, enhanced by 457.9 nm excitation, are nearly equal in both ferredoxins. These data were used to demonstrate the presence of hybrid clusters [4Fe-(4-n)S-nSe] (n = 1, 2, 3) in a ferredoxin the active sites of which had been reconstituted in the presence of both S and Se.
| S-EPMC1153550 | biostudies-other
Project description:The mechanism of the enzymic reaction of an iron-containing superoxide dismutase purified from the marine bacterium Photobacterium leiognathi was studied by using pulse radiolysis. Measurements of activity were done with two different preparations of enzyme containing either 1.6 or 1.15 g-atom of iron/mol. In both cases, identical values of the second-order rate constant for reaction between superoxide dismutase and the superoxide ion in the pH range 6.2-9.0 (k=5.5 X 10(8) M-1-S-1 at pH 8.0) were found. As with the bovine erythrocuprein, there was no evidence for substrate saturation. The effects of reducing agents (H2O2, sodium ascorbate or CO2 radicals) on the visible and the electron-paramagnetic-resonance spectra of the superoxide dismutase containing 1.6 g-atom of ferric iron/mol indicate that this enzyme contains two different types of iron. Turnover experiments demonstrate that only that fraction of the ferric iron that is reduced by H2O2 is involved in the catalysis, being alternately oxidized and reduced by O2; both the oxidation and the reduction steps have a rate constant equal to that measured under turnover conditions. These results are interpreted by assuming that the superoxide dismutase isolated from the organism contains 1 g-atom of catalytic iron/mol and a variable amount of non-catalytic iron. This interpretation is discused in relation to the stoicheiometry reported for iron-containing superoxide dismutases prepared from several other organisms.
| S-EPMC1164466 | biostudies-other
Project description:Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3(•), CO3(•-),(•)NO2 and GS(•)). The rate constants for the Amplex® Red oxidation by N3(•) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(•-) ((2)k=7.6·10(8)M(-1)s(-1), at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.
| S-EPMC5697983 | biostudies-literature
Project description:1. Detailed studies on the mechanism of the enzymic reaction of bovine superoxide dismutase were carried out by using pulse radiolysis and electron paramagnetic resonance (e.p.r.). 2. The second-order rate constant for reaction between superoxide dismutase and the superoxide ion was redetermined as (2.37+/-0.18)x10(9)m(-1).s(-1) at 25 degrees C. This reaction governs the turnover, and any first-order steps must have rate constants higher than about 10(6)s(-1). Turnover has a low activation energy and is slowed substantially when the viscosity is increased with glycerol, confirming that the reaction rate is near the limit for diffusion control. In water a reversible conformation change to a less active form appears to take place above about 40 degrees C. 3. Pre-steady-state rates of reduction and reoxidation of copper in the enzyme are consistent with these processes being rate-limiting in enzyme turnover. 4. Examination, with the help of computer simulation, of the e.p.r. spectra at 9 and 35GHz of native superoxide dismutase indicated that, apart from 10-20% of impurities, only one species of Cu(2+) is distinguishable. Further, the specific activity of our enzyme preparations, measured by pulse radiolysis, is at least as high as that obtained by other workers. 5. Nevertheless, measurement of the proportion of copper present as Cu(2+) (determined both optically and by e.p.r. spectroscopy) in the steady states approached from both the oxidized and the reduced forms of the enzyme, indicates (after allowing for the impurities) that only half of the copper atoms participate in turnover. E.p.r. spectroscopy provided no evidence for differences between functioning and non-functioning Cu(2+) atoms. 6. It is suggested that the results may be best interpreted in terms of an allosteric type of mechanism, with two initially indistinguishable copper atoms in the enzyme. Reaction of one of these with a superoxide ion then renders the other, at least transiently, unreactive.
| S-EPMC1166250 | biostudies-other
Project description:Reactions of the [3Fe-4S] cluster and various metallated [M3Fe-4S] adducts co-ordinated in the ferredoxin from the hyperthermophile Pyrococcus furiosus have been studied by protein-film voltammetry, bulk-solution voltammetry, solution kinetics and magnetic CD (MCD). The [3Fe-4S] cluster exhibits two couples, [3Fe-4S]+/0 and [3Fe-4S]0/2-. Film voltammetry is possible over a wide pH range (2-8), revealing that the [3Fe-4S]+/0 couple shows a complex pH dependence with pKred1=2.8, pKox=4.9 and pKred2=6.7. From MCD, pKred1 corresponds with protonation of [3Fe-4S]0 to give a spectroscopically distinct species, as reported for ferredoxins from Azotobacter and Sulfolobus. The status of the disulphide/disulphydryl entity makes no significant difference to the data (given for the -S-S- form). Formation of the hyper-reduced [3Fe-4S]2- state is observed, requiring 3H+ for the overall 3e- reduction of [3Fe-4S]+, the change therefore being electroneutral. By comparison with the ferredoxin from Desulfovibrio africanus, uptake of Fe(II) and other M(II) by [3Fe-4S]0 to give [M3Fe-4S] clusters is slow (t1/2>10 min at room temperature, slower still if the protein is adsorbed on the electrode), whereas reaction with Tl(I) to produce [Tl3Fe-4S] is very rapid (t1/2<<1 s), suggesting that co-ordination of Tl does not require reorganization of the protein structure. Rates of formation of [3Fe-4S] from [M3Fe-4S] adducts increase sharply at high potentials, showing that metal release involves a labile 'super-oxidized' [M3Fe-4S]3+ state.
| S-EPMC1219790 | biostudies-other
Project description:The air- and moisture-stable iron-sulfur carbonyl clusters Fe3S2(CO)7(dppm) (1) and Fe3S2(CO)7(dppf) (2) carrying the bisphosphine ligands bis(diphenylphosphanyl)methane (dppm) and 1,1'-bis(diphenylphosphanyl)ferrocene (dppf) were prepared and fully characterized. Two alternative synthetic routes based on different thionation reactions of triiron dodecacarbonyl were tested. The molecular structures of the methylene-bridged compound 1 and the ferrocene-functionalized derivative 2 were determined by single-crystal X-ray diffraction. The catalytic reactivity of the trinuclear iron-sulfur cluster core for proton reduction in solution at low overpotential was demonstrated. These deeply colored bisphosphine-bridged sulfur-capped iron carbonyl systems are discussed as promising candidates for the development of new bioinspired model compounds of iron-based hydrogenases.
| S-EPMC4612652 | biostudies-literature
Project description: Not available
| S-EPMC4866492 | biostudies-literature
Project description:DNA nanotubes are cylinder-like structures formed from DNA double-helical molecules whose helix axes are fused at least twice by crossovers. It is potentially useful to use such tubes as sheaths around rodlike species that arise in biological systems and in nanotechnology. It seems easiest to obtain such sheathing by joining two or more components around an object rather than attempting to thread the object through a cavity in the tube. We report two examples of tubes containing a specific number of helices that are assembled from half-tube components. These tubes are a six-helix bundle and an eight-helix bundle, constructed respectively from two bent triple-crossover (BTX) molecules and from two four-helix arched motifs. Both species contain single strands in one molecule that are missing in its mate. The six-helix bundle is formed from two different BTX molecules, whereas the eight-helix species is a closed cyclic dimer of the same molecule. We demonstrate the formation of these species by gel electrophoresis, and we examine their arrangement into long one-dimensional arrays by means of atomic force microscopy.
| S-EPMC2527457 | biostudies-literature